To date very limited knowledge is available on 3-ketosteroid 9α-hydroxylase (KSH), the enzyme performing the 9α-hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in microbial sterol/steroid degradation. No nucleotide sequences of the genes encoding KSH components have been reported. Furthermore, difficulties are faced during enzyme purification procedures (Chang, F. N. et al. Biochemistry (1964) 3:1551–1557; Strijewski, A. Eur. J. Biochem. (1982) 128:125–135). A three-component monooxygenase with KSH activity has been partially purified from Nocardia sp. M117 and was found to constitute a three-component enzyme system, composed of a flavoprotein reductase and two ferredoxin proteins (Strijewski, A. Eur. J. Biochem. (1982) 128:125–135). In Arthrobacter oxydans 317, 9α-hydroxylation of the steroid poly-cyclic ring structure appeared plasmid-borne (Dutta, R. K. et al. J. Basic Microbiol. (1992) 32:317–324). Nucleotide sequence analysis of the plasmid, however, was not reported.
The lack of genetic data has hampered the construction of molecularly defined mutant strains with desired properties (i.e. blocked 9α-hydroxylation of steroids) by genetic engineering. Mutants have been isolated by classical mutagenesis, but these strains usually are inadequate in industrial processes mostly due to genetic instability and/or low bioconversion efficiencies. Molecularly defined mutants have advantages compared to mutants generated by classical mutagenesis. The constructed mutants are genetically stable and the introduced mutations are well-defined genetic modifications. Construction of genetically engineered strains make the widespread use of chemical agents to block 9α-hydroxylation (e.g. α,α-dipyridyl, o-phenanthroline) obsolete. Chemical agents used to block KSH activity mostly are not reaction specific and inhibit other important enzymatic reactions (e.g. sterol 26-hydroxylation in sterol side chain degradation), which may have negative effects on sterol bioconversion efficiency. The use of defined mutants by genetic engineering overcomes these problems.
3-Ketosteroid 9α-hydroxylase (KSH) is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the conversion of AD into 9α-hydroxy-4-androstene-3,17-dione (9OHAD) and ADD into the chemically unstable compound [9OHADD]. KSH activity has been found in many bacterial genera (Martin, C. K. A. Adv. Appl. Microbiol. (1977) 22: 29–58; Kieslich, K. J Basic Microbiol. (1985)25: 461–474; Mahato, S. B. et al. Steroids (1997)62: 332–345): e.g. Rhodococcus (Datcheva, V. K. et al. Steroids (1989) 54:271–286; Van der Geize et al. FEMS Microbiol. Lett. (2001) 205: 197–202, Nocardia (Strijewski, A. Eur. J. Biochem. (1982) 128:125–135), Arthrobacter (Dutta, R. K. et al. J. Basic Microbiol. (1992)32:317–324) and Mycobacterium (Wovcha, M. G. et al. Biochim Biophys Acta (1978) 531:308–321). Bacterial strains lacking KSH activity are being considered important in sterol/steroid biotransformation. Mutants blocked in KSH activity will be able to perform only the KSTD (3-ketosteroid Δ1-dehydrogenase) reaction, thereby allowing selective Δ1-dehydrogenation of steroid compounds. Examples are the cortisol biotransformation into prednisolone and the AD biotransformation into ADD. Sterol bioconversion by mutants blocked at the level of steroid 9α-hydroxylation may also carry out a selective degradation of the sterol side chain thereby accumulating AD and/or ADD which are excellent precursors for the synthesis of bioactive steroid hormones.